KR101027082B1 - Streptomyces misonensis CJS-70 with antibacterial activity to plant pathogens and microbial agent for controlling plant pathogens using the same - Google Patents

Abstract

The present invention is Streptomyces misonensis CJS-70 strain having antibacterial activity against plant pathogens (Streptomyces misonensis) CJS-70; KCTC 11110BP) and a microbial agent for controlling plant pathogens using the same, and more particularly, a plant causing gray mold, hairy rot, scab, root rot, etc. using the strain itself, mycelia of the strain, spores or mixtures thereof. The present invention relates to a microbial agent that can control pathogenic bacteria.

Streptomyces misonensis CJS-70, microbial preparation, ash fungus, seedling disease, spot disease, root rot

Description

Streptomyces misonensis CJS-70 with antibacterial activity to plant pathogens and microbial agent for controlling plant pathogens using the same

The present invention relates to a Streptomyces misonensis CJS-70 strain having an antimicrobial activity against plant pathogens and a microbial agent for controlling plant pathogens using the same, and more particularly, the strain itself, mycelia, spores or mixtures thereof. It relates to a microbial agent that can control plant pathogenic bacteria causing gray mold, hairy rot, spots, root rot and the like.

Crop diseases caused by phytopathogenic fungi are a very limiting factor in the stable production of crops along with pests and weeds. Among many phytopathogenic fungi, fungal nuclei, such as Botrytis cineraria, are mostly infectious soils, causing massive damage to various crops.

Since these plant pathogenic fungi exist in fungal conditions and survive in harsh environments such as high temperature and low temperature, these fungal nuclei are known to be the primary source of infection for plants every year.

Such fungal nuclei are difficult to expect effective control because they can survive in poor environmental conditions in diseased tissues or soil and are resistant to degradation and spores or inhibition by neighboring antagonistic microorganisms than spores or mycelium forms.

Methods for controlling such fungi include physical methods to improve the cultivation environment, chemical methods using fungicides, and microbiological methods using microorganisms. However, physical control alone cannot be expected to have a great effect, and chemical control has not only significantly reduced the control effect on the packaging due to the emergence of drug-resistant pathogens, but also leads to serious environmental pollution by spraying a large amount of chemical pesticides. In addition, the use of chemically synthesized pesticides is increasingly regulated due to toxicity to humans and animals, residual toxicity in soil and crops, and environmental pollution due to packaging containers. Its use is greatly limited due to the strengthening of specific regulations on pesticides.

Therefore, in recent years, research on microbiological control has been actively conducted as an alternative method, and the development of effective control methods for environmentally friendly agriculture is urgently required.

As an environmentally safe and effective control method, biological control using a microbial agent is important. Since the early 1970s, many studies have been conducted by many researchers at home and abroad. Recently, more than 10 kinds of microbial products have been commercialized in the world, and research on the development of microbial products is rapidly increasing due to a change in social awareness aiming for environmentally safe and sustainable agriculture.

Typical microbial agent developed and commercialized in a foreign country is currently tricot der Do not annuum (Trichoderma harzianum) and tricot der Matthew Polis Forum (T. polysporum) made a BINAB T (1988, ER Butts International ) to inhibit Wood rot fungi, writing GlioGard (1990, WR Grace Co.), a bottle control agent for greenhouse flower seedlings using Gliocladium virens , Fstop (1991, Kodak), Trichodex (1997, Israel) Developed by KODIAK (1990, Gustafson) and Pseudomonas fluorescens using seed antagonist Bacillus subtilis) to control cotton lipotonia and Pythium sp. DAGGER (1990, Ecogen Inc.), Pseudomonas Sefar Asia (P. cepacia) for BLUE CIRCLE the seed treatment of multiple crops using (1990, Stine Microbial Products), and antagonistic actinomycetes host Sat My process and the like so Seo irregularities disk (Streptomyces griseoviridis) the Fusarium (Fusarium) wilt, Mo jalrokbyeong control agent MYCOSTOP (1985, Kemira Oy Biocontrol of Finland) using.

In Korea, research on biological control of ginseng root rot has been conducted since the early 1970s, and studies on biological control of red pepper disease, cucumber strawberry wilting disease, sesame seedling disease, and ash fungus have been conducted, but the results are insignificant.

In addition, microorganism pesticides are microorganisms for fungicides, and bacteria include Bacillus sp. BS555, KCTC 8821P, Bacillus polymoxa B36, FERM BP-6068, Pseudomonas aureofaciens B5, FERM BP- 6067), Enterobacter claw dehydratase (Enterobacter cloacae B51, FERM BP-6069) and Bom1 (KFCC 10992) and Bom2 (KFCC 10993) in Streptomyces, an actinomycetes that secrete antifungal substances.

Furthermore, the microorganisms having antagonistic ability of the pathogenic fungus of Bacillus subtilis bar amyl duck keupa City Enschede (Bacillus subtilis var. Amyloliquefaciens KL114, KCTC 8913P), Bacillus amyl Lori query Patience (Bacillus amyloliquefaciens KTGB0202 KCTC 10564BP), alkaline Jeans wave Alcaligenes faecalis KL1179, KCTC 8914P), Pseudomonas Pseudomonas aeruginosa KL1121, KCTC 8915P), Pseudomonas aurantiaca KL1326, KCTC 8916P), Bacillus subtilis and Pseudomonas fluorescences, and Pseudomonas fluorescence bios 1 (Biovar 1), which have antagonistic ability against actinomycetes Streptomyces genus, and fungi Del Mar not ahnyum, Penny chamber in Solarium (Penicillium sp.), in Aspergillus (Aspergillus sp.), redundancy parasitic fungi in Acre help monitor the streak lithium BCP (Acremonium strictum, KCTC 0639BP), etc. is under study.

The principle of biological control by antagonistic microorganisms is to reduce the density or pathogenesis of pathogens through antibiotics produced by antibiotics, parasitic or phagocytosis by cell wall degrading enzymes, and nutrient competition or niche competition. The main mechanisms of action are cross-protection and induction of host resistance using non-pathogenic or weakly-pathogenic strains. For example, the inhibitory action of fungal diseases caused by antibiotics or volatile substances, toxins, and special inhibitors secreted by the genus Trichoderma and gliocladium is known.

On the other hand, antagonism by parasitic or phagocytosis has been actively studied in recent years. Pathogens caused by chitinase, glucanase, and cellulase, which are cell wall degrading enzymes secreted by antagonistic bacteria Dissolution of is known as the main mechanism of action.

The purpose of the present invention has been reported a variety of microbial pesticides having an antagonistic action against plant pathogenic bacteria as described above, but to provide a more effective and environmentally friendly microbial agent, for this purpose a new strain having antimicrobial activity against plant pathogens It is to seek more eco-friendly agriculture using new strains.

To solve the above problems,

The present invention provides a Streptomyces misonensis CJS-70 strain having antagonistic ability against plant pathogens.

In another aspect, the present invention provides a microbial agent for controlling plant pathogens containing the culture medium of Streptomyces misonensis CJS-70 strain as an active ingredient.

Novel microorganisms and microbial control agents using the same according to the present invention, Botrytis cinerea which causes gray mold disease, Pythium ultimum which causes hairy ulcer disease, Alternaria panax, Fusarium sp., Which causes root rot, Cylindrocarpon destructans, Rhizoctonia solani, and pepper blight Not only does it have a good inhibitory effect on the causative Phytophthora capsici and the streptomyces scabies that cause potato beetle disease, but also when used as an environmentally friendly pollution-free pesticide It can be used in pesticide industry and environmental industry because it can solve the destruction and shrinkage toxicity problem.

The present invention is Streptomyces misonensis CJS-70 (Streptomyces misonensis) having the ability to antagonize plant pathogens CJS-70; KCTC 11110BP) strain.

In the present invention, the plant pathogens, Botrytis Ceria, Pisium Ultimum, Alternaria Panax, Fusarium Solani, Cylindrocarpon Destructans, Laichetonia Solani, Phytopsofa capsaish and Streptococcus At least one selected from the group consisting of Maises Skabiss is preferred, but is not limited thereto.

In addition, in the present invention, the plant pathogen, Botrytis cineraria that causes gray mold; Pisium Ultimum, the causative agent of parental disease; Alternaria panax, which causes spots disease; Fusarium Solani, Cylindrocarpon Destructans, Raiztonia Solani causing root rot; Phytoprosora capsaish, which causes pepper blight; And at least one selected from the group consisting of Streptomyces scabies, which cause potato beetle disease, but is not limited thereto.

The present invention provides a microbial preparation for controlling plant pathogens containing the culture medium of the Streptomyces misonensis CJS-70 strain as an active ingredient.

In the present invention, the culture solution may be diluted 0-500 times after growth and growth of the Streptomyces misonensis CJS-70 strain in the culture medium.

In addition, the culture medium in the present invention may be used G.S.S medium (glucose-soybean meal-starch medium).

In addition, the culture medium in the present invention may include one or more selected from the group consisting of the hyphae, spores and mixtures thereof of the Streptomyces misonensis CJS-70 strain.

In the microorganism preparations for controlling the plant pathogens of the present invention,

The plant pathogens include Botrytis Ceria, Pisium Ultimum, Alternaria Panax, Fusarium Solani, Cylindrocarpon Destructans, Raiztonia Solani, Phytopsofa capsaish and Streptomyces scar At least one selected from the group consisting of BS is preferred, but is not limited thereto.

In addition, the plant pathogens, Botrytis cineraria that causes gray mold; Pisium Ultimum, the causative agent of parental disease; Alternaria panax, which causes spots disease; Fusarium Solani, Cylindrocarpon Destructans, Raiztonia Solani causing root rot; Phytoprosora capsaish, which causes pepper blight; And at least one selected from the group consisting of Streptomyces scabies, which cause potato beetle disease, but is not limited thereto.

Hereinafter, the present invention will be described in more detail.

The novel Streptomyces misonensis strain according to the present invention is a botrytis cineria, fishium Ultimum, Alternaria panax, fusarium solanie, silindrocapon destract in soil with good internal parts in Jeongeup, Jeollabuk-do It is a newly discovered strain as a result of selecting and isolating and identifying microorganisms having a strong antibacterial activity in common in Tans, Lysatonia solani, Phytopsora capsaish and Streptomyces scabies. The new strain was named Streptomyces misonensis CJS-70, and was deposited with the Korea Biotechnology Research Institute Gene Bank on April 9, 2007. The accession number is KCTC 11110BP.

As a result of analyzing the antimicrobial activity spectrum of the novel Streptomyces misonensis CJS-70 strain of the present invention, it was confirmed that there is a strong antimicrobial activity against the above-mentioned plant pathogens, and the antimicrobial activity according to the degree of dilution of the new strain was also revealed by an experiment. .

In addition, in the present invention, the culture medium and temperature conditions for the mass production of Streptomyces misonensis CJS-70 strain having anti-plant pathogen activity can be more effectively produced.

The present invention includes biopesticides such as microbial preparations for controlling plant pathogens containing the culture medium of the Streptomyces misonensis CJS-70 strain as an active ingredient.

Such biopesticides may be prepared using, in addition to the Streptomyces misonensis CJS-70 cells themselves, mycelia, spores, or mixtures thereof, carriers usable as pesticide preparations. As the carrier, any acceptable carrier may be used as a conventionally used pesticide formulation, but it is particularly preferable to use a cryopreservative, skim milk, yeast extract, and the like.

The active ingredient (culture liquid) according to the present invention is used by diluting 0 to 1000 times with purified water, and preferably diluted 0 to 500 times to form a powder. The microbial control agent thus prepared is preferably treated with a foliage treatment method on the plant's ground from 10 days after planting or just before the onset of the crop in order to control gray mold, hairy greens, spots, root rot and the like.

The microbial agent may be used as a pesticide ingredient such as seed coating agent, microbial nutritional agent, soil improving agent, etc. in addition to microbial control agent for controlling plant pathogens.

Hereinafter, the present invention will be described in detail with reference to Examples. However, the following examples do not limit the technical scope of the present invention.

Example 1 Isolation and Identification of Strains

1. Isolation of Strains

In this example, Botrytis cieria, Pisium Ultimum, and Fusarium genus as plant pathogenic experimental strains, soil and compost samples were collected from all over the country in order to isolate the antagonistic microorganisms.

1 g of the collected sample was mixed with 10 mL of sterile water, shaken and incubated at 28 ° C. for 30 minutes, and then diluted to 10,000 times by soil dilution agar plate method, and 0.1 mL of the diluted solution was prepared using GSS medium (glucose-soybean meal-starch medium) ( See Table 1), evenly spread on the entire surface of the medium, and then cultured for 5 days at 28 ℃ incubator, a single colony of fungi (single colony) was purely separated and transferred to GSS slope medium and stored at 4 ℃.

Streptomyces misonensis obtained in the present invention exhibits a strong antimicrobial activity in common in Botrytis Ceria, Pisium Ultimum and Fusarium, and selected one strain having the best antimicrobial activity.

TABLE 1 G.S.S medium composition table

Badge Ingredient Concentration (g / L) Starch 10 Glucose 20 Soy flour 25 Beef Extract One Yeast extract 4 Sodium chloride 2 Potassium phosphate 0.25 Calcium carbonate 2 pH 7.2

2. Identification of Strains

Identification of the strains selected above was carried out as follows.

1) Morphological characteristics

Selected strains were inoculated in G.S.S agar medium and incubated at 28 ° C. for 5 days. Observations produced light grayish brown or yellow mycelia depending on the type of medium. The production of the pigment is characterized by the presence or absence of production depending on the medium used, detailed cultural and physiological characteristics are shown in Table 2 below.

<Table 2> Culture Characterization of Selected Strains

 Analysis item characteristic Spore color / hyphae color by badge type ISP 2 badge Taupe / yellow ISP 3 badge Taupe / brown ISP 4 badge Taupe / brown ISP 5 badge Taupe / yellow Spore chain morphology gyre Spore Count / Spore Chain 10-50 pieces Whether pigments are produced ISP 2 badge U ISP 3 badge radish ISP 4 badge radish ISP 5 badge U Carbon use Arabinose - Fructose + Glucose + Mannitol + Raffinose - Sucrose - Xylose + Whether to grow when the following ingredients are added 5% (w / v) sodium chloride (NaCl) - 100 μg / ml thallium acetate - 1 μg / ml crystal violet + 20 μg / ml streptomycin - 10 μg / ml penicillin - Lowest pH for growth 3.5

(+: Possible,-: impossible)

2) 16S rDNA sequence analysis

In order to confirm the phylogenetic location of the selected strains, 446 bp sequences corresponding to more than 29% of the 16S rDNA sequences were analyzed. PCR amplification of 16S rDNA fragment 5'-AGA GTT TGA TCM TGG CTC AG-3 'from the selected strain with a primer set, and then amplified product was purified and inserted into TOPO TA cloning kit (Introtrogen). E. coli was transformed. After the plasmid was extracted from the transformed E. coli, the nucleotide sequence was analyzed using an Applied Biosystems model 373A automatic sequencer and a kit (BigDye Terminator Cycle Sequencing kit, Perkin-Elmer Applied Biosystems). 16S rDNA of the selected strain is shown in Figure 1 the results of homology analysis using the blast program of the gene bank.

Referring to FIG. 1, as a result, the selected strain exhibits 99% homology with Streptomyces misonensis NRRL B-3230, and thus, the strain of the present invention was finally identified as a Streptomyces misonensis strain. Therefore, on April 9, 2007, it was deposited as a patent strain to the Korea Biotechnology Research Institute Gene Bank and was assigned accession number KCTC 11110BP.

Example 2 Antagonistic Assay for Various Plant Pathogens

Plant pathogens include a variety of pathogens that cause enormous damage to plants, including Botryzinis cinemaia, Pisium Ultimum, Alternaria Panax, Fusarium Solani, Cylindrocarpon destructans, Phytoprosola capsaish, and lye. Eight test strains of Jotonia Solani and Streptomyces scabies were distributed at the Korea Agricultural Microbial Resources Center (KACC) to assay the antagonism of Streptomyces misonensis CJS-70 strain.

After sterilizing potato agar medium (PDA, Difco), plate and mix 200 μl of the culture medium containing mycelia and spores of each pathogen in a medium at 50 ° C. (8 mm in diameter) and then placed in a petri dish mixed with pathogens and incubated for 3 to 4 days at 28 ℃ to grow various pathogens well.

The effect of antagonistic microorganisms on various pathogens was investigated with a low support diameter, which is shown in Table 3 below.

As can be seen in Table 3, as a result of the antagonistic effect of the antagonistic microorganism Streptomyces misonensis CJS-70 strain of the present invention and plant pathogens, the strain of the present invention is a causative agent of gray mold It showed excellent inhibitory effects against Tritis cineraria, Alternaria panax, which causes spots, Fusarium, which causes root rot, Cylindrocarpon destructans, and Lysatonia solani. It was confirmed that the inhibitory effect was also exhibited against pithyum ultimum, phytoprosora capsaici causing causal pepper plague, and streptomyces scavies, which causes cabbage.

Table 3 Antimicrobial Activity of Streptomyces Misonensis CJS-70 Strain against Pathogenic Assay

Assay test bacteria Activity level Botrytis Ceria cineria KACC No.40573) ++++ Cylindro car phone destructans
( Cylindrocarpon destructans KACC No.41077) +++ Fusarium solani f. sp.pisi KACC No.42181) +++ Phytopthora capsici KACC No.40475) +++ Pythium ultimum KACC No.40705) +++ Alternaria panax KACC No.42141) +++ Rhizoctonia solani KACC No. 40123) ++ Streptomyces scabies ) +++ ++++: size of clear zone> 15 mm,
+++: 10-15 mm,
++: 5-10 mm,
+: 1 to 5 mm,
-: No activity

Streptomyces misonensis CJS-70 strain of the present invention showed excellent antagonism against most of the experimental strains, Figures 3 to 10 as evidence that each streptoto using agar diffusion method (agar diffusion method) It was a result showing the antimicrobial activity of the Myses misonensis CJS-70 strain.

Example 3 Antagonistic Assay According to Dilution of Streptomyces Misonensis CJS-70 Strain Culture

After culturing the Streptomyces misonensis CJS-70 strain under the conditions of Example 2 and diluting the culture medium at a constant rate, it is reported that the cause of gray mold according to the degree of dilution of the culture medium in the same manner as in Example 2 The results of measuring antimicrobial activity against Tritis cieria, Pisium Ultimum, which causes Mozzarlock disease, and Fusarium Solani, which causes root rot, are shown in Table 4 below.

<Table 4> Antimicrobial Activity According to Dilution of Streptomyces Misonensis CJS-70 Strain Culture

Test bacteria
Dilution factor Botrytis Cinema Pisium Ultimum Fusarium Solani Stock solution ++++ +++ +++ 2-fold dilution ++++ +++ +++ 5-fold dilution ++++ +++ +++ 10-fold dilution ++++ +++ +++ 20-fold dilution +++ ++ ++ 50-fold dilution +++ ++ ++ 100-fold dilution +++ ++ + 200-fold dilution ++ + + 500-fold dilution + + - 1000-fold dilution + - -

++++: Clear zone size> 15 mm, +++: 10 ~ 15 mm, ++: 5 ~ 10 mm, +: 1 ~ 5 mm,-: No activity

Example 4 Mass Culture of Streptomyces Misonensis CJS-70 Strain

Cultivation of the antagonistic microorganism Streptomyces misonensis strains was cultivated in a 2.5 L fermentor using a medium commonly used for culturing Streptomyces spp. The medium composition used was starch 5 g, glucose 20 g, soybean powder 25 g, beef extract 1 g, yeast extract 4 g, sodium chloride 2 g, potassium phosphate 0.25 g, calcium carbonate 2 g, dissolved in 1 L of distilled water and then pH Was adjusted to 7.2 to prepare and use a conventional liquid medium.

Species culture was incubated in a 500 ml Erlenmeyer flask using a 100 ml liquid medium in which yeast extract 5 g / L, beef extract 5 g / L and glucose 10 g / L were added. Culture conditions were shaken at 120 rpm for 120 hours at 28 ℃, 5% (v / v) of the culture medium containing Streptomyces misonensis inoculated in a 2.5 L fermentor containing 1.5 L production medium and 28 ℃ Incubated for 120 hours at 200 rpm and 1 vvm.

The present invention is a microbial control agent having an antagonistic resistance to plant pathogenic bacteria as salping, and serves as an environmentally friendly pesticide, it is possible to mass-produce microbial preparations for controlling plant pathogens by mass cultivation of new strains and income of farmers It will be able to contribute to the development of national industry by raising the level of income and ensuring the safe consumption of agricultural products.

1 is a analysis of 446 bp of the new strain Streptomyces misonensis 16S rDNA nucleotide sequence of the present invention.

2 is an electron micrograph taken at 10,000 times magnification of the new strain Streptomyces misonensis of the present invention.

Figure 3 shows the antimicrobial activity of the novel strain Streptomyces misonensis of the present invention against Botrytis cinerea.

Figure 4 shows the antimicrobial activity of the new strain Streptomyces misonensis of the present invention against Pythium ultimum.

Figure 5 shows the antimicrobial activity of the new strain Streptomyces misonensis of the present invention against Streptomyces scabies.

Figure 6 shows the antimicrobial activity of the new strain Streptomyces misonensis of the present invention against Fusarium solani.

Figure 7 shows the antimicrobial activity of the new strain Streptomyces misonensis of the present invention against Cyclodrocabon destractans.

Figure 8 shows the antimicrobial activity of the new strain Streptomyces misonensis of the present invention against Alternaria panax.

Figure 9 shows the antimicrobial activity of the new strain Streptomyces misonensis of the present invention against Rhizoctonia solani.

Figure 10 shows the antimicrobial activity of the novel strain Streptomyces misonensis of the present invention against Phytophthora capsici.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Streptomyces misonensis CJS-70 with antibacterial activity to          plant pathogens and microbial agent for controlling plant          pathogens using the same <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 446 <212> DNA <213> Streptomyces misonensis CJS-70 <400> 1 ctcttctgca ggtaccgtca ctttcgcttc ttccctgctg aaagaggttt acaacccgaa 60 ggccgtcatc cctcacgcgg cgtcgctgca tcaggctttc gcccattgtg caatattccc 120 cactgctgcc tcccgtagga gtctgggccg tgtctcagtc ccagtgtggc cggtcgccct 180 ctcaggccgg ctacccgtcg tcgccttggt gagccgttac ctcaccaaca agctgatagg 240 ccgcgggctc atcctgcacc gccggagctt tacaccgtca aggatgccca agacggtcat 300 atccggtatt agaccccgtt tccagggctt gtcccagagt gcagggcaga ttgcccacgt 360 gttactcacc cgttcgccac taatccaccc cgaagggctt catcgttcga cttgcatgtg 420 ttaagcacgc cgccagcgtt cgtcct 446  

Claims (7)

Streptomyces misonensis CJS-70 (KCTC 11110BP) strain characterized by antagonistic ability to Botrytis cineraria that causes gray mold. delete delete Streptomyces misonensis CJS-70 CJS-70; KCTC 11110BP) microorganism for control of botrytis cineraria, which is a causative agent of gray mold, which contains as an active ingredient culture medium diluted 0-500 times after growing and growing in GSS medium (glucose-soybean meal-starch medium) Formulation. delete delete delete

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